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iTRAQ-based quantitative proteomic analysis of germinating barley under GA and ABA treatments Yuqing Huang, Shengguan Cai, Jianbin Zeng, Dezhi Wu, and guoping Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04865 • Publication Date (Web): 21 Feb 2017 Downloaded from http://pubs.acs.org on February 22, 2017
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Journal of Agricultural and Food Chemistry
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iTRAQ-based quantitative proteomic analysis of germinating barley under
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GA and ABA treatments
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Yuqing Huang, Shengguan Cai, Jianbin Zeng, Dezhi Wu, Guoping Zhang*
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Department of Agronomy, Zhejiang Key Lab of Crop Germplasm, Zhejiang University,
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Hangzhou 310058, China
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*
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571 88982117).
Corresponding author (E-mail:
[email protected], phone: +86 571 88982115, Fax: +86
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Key words: barley, gibberellin (GA), abscisic acid (ABA), germination, proteomics
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Abstract
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The degradation of starch in barley grains is a primary step of beer production. The addition
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of appropriate amount of GA promotes the production of fermentable sugars, beneficial to
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brewing industry. However, the response of proteomics in germinating barley to GA and
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ABA treatments is not thoroughly understood. In this study, iTRAQ (isobaric tags for relative
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and absolute quantitation) proteomics analysis was performed to illustrate the change of
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proteins in Tibetan wild barley XZ72 and XZ95 under GA and ABA treatments during
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germination. XZ72 had more proteins up-regulated than XZ95 under GA treatment, while
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under ABA treatments, XZ95 had more proteins up-regulated than XZ72. Concerning the
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proteins involved in energy metabolism under GA treatment, XZ72 had more proteins up-
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regulated than XZ95. Among the 174 proteins related to starch metabolism, 31 proteins
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related to starch hydrolysis, such as α-amylase, α-glucosidase and β-fructofuranosidase,
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showed higher relative abundance in control and GA treatment in XZ72 than in XZ95.
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Analysis of correlation between proteins and metabolites indicated that higher hydrolases
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activity is beneficial for the accumulation of fermentable sugars during germination. On the
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other hand, 26 starch synthesis-related proteins were up-regulated in XZ95 under ABA
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treatment. It may be suggested that GA-induced proteins act as accelerators of starch
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degradation while ABA-induced proteins inhibit starch degradation. The current results
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showed that XZ72 is highly capable of allocating the starch hydrolysing enzymes which play
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important roles in starch breakdown.
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1. INTRODUCTION
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Malting is the first step in brewing industry, and its process consists of optimized grain
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germination when hydrolysis enzymes are secreted from the aleurone layer and the starch is
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digested into fermentable sugars.1 The hydrolysis of starch to the fermentable carbohydrates
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is an important step of brewing, which is achieved by the concerted function of α-amylase, β-
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amylase, limit dextrinase and α-glucosidase.2 Starch is the main carbohydrate reserve in
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cereal seeds. The starch composition in barley (Hordeum vulgare L.) grains is around 70-75%
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amylopectin and 25-30% amylose.3
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Gibberellins (GA) and abscisic acid (ABA) are two most common hormones which
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function as regulators of plant development. Many studies on a wide variety of plants have
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shown that GA and ABA have a strong influence on seed dormancy and germination.4,5 The
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increase of ABA concentration leads to the accumulation of storage nutrients and seed
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dormancy.6 On the contrary, GA breaks seed dormancy and promotes germination.7 The
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balance of GA and ABA controls the shift between maturation and germination. During
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germination, GA is synthesized in the embryo and transferred to the aleurone layer, where it
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stimulates the production and development of hydrolytic enzymes. The enzymes are further
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transferred to the endosperm to catalyze the hydrolysis of starch.
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It is a common practice in the brewing process to add an appropriate dose of GA to
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germinating barley seeds so as to enhance both α-amylase and limit dextrinase (LD),
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thusaccelerating germination.8 The mechanism for induced expression of α-amylase genes by
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GA has been extensively studied. GA stimulates while ABA inhibits the synthesis of α-
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amylase by regulating the mRNA level. The transcription factor GAMYB, which regulates
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the expression of α-amylase gene was highly induced by the addition of GA. On the other
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hand, GA3-induced transcription of the α-amylase encoding genes was inhibited by ABA.9
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Although the functional and expressional analysis of the genes related to α–amylase has been
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well established, the changes of β-amylase, limit dextrinase and α-glucosidase during
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germination have not been thoroughly understood.
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Tibetan wild barley is a progenitor of cultivated barley and rich in genetic diversity.10 As
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shown in previous studies, wild barley displays higher variation in limit dextrinase inhibitor,
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β-amylase activity and phenolic acids.11-15 According to our previous results, the malting
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quality of XZ72 and XZ95 differed greatly.16 Application of GA increased sugars and amino
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acids concentrations of germinating barley grains, with more increase in XZ72 than in
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XZ95.16 More fermentable sugars result in higher yield of malt extract, which is beneficial for
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beer production. However, the underlying mechanism in the difference of these metabolites
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between XZ72 and XZ95 is still unclear.
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The ‘omic’ strategy, such as proteome and metabolome analysis, has been considered as
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powerful tools in analyzing complex biological concerns.17 Actually metabolome,
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transcriptome and proteome methods have been widely applied in the studies of seed
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germination.18,19 Jin et al.20 used two dimensional electrophoresis to analyze the protein
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difference between two malt barley genotypes with distinct filterability and identified 40
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proteins, most of which were hydrolases and pathogen-related proteins.20 The iTRAQ is a
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more powerful and sensitive approach for protein quantification on the basis of peptide
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labelling and quantification. With the capacity of up to 8 samples analyzed simultaneously,21
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it improves largely the throughput of 2D-gel based techniques.22 Although the iTRAQ-based
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proteomics has been widely applied in studies of abiotic stress23,24 and nutrient deficiency
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responses25, little research has been done on grain quality, including barley malt quality. In
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this study, we conducted iTRAQ analysis to identify the proteins responding to GA and ABA
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treatments during germination using two barley genotypes differing in malt quality.
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Journal of Agricultural and Food Chemistry
2. MATERIALS AND METHODS
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2.1. Malting procedure. Two Tibetan wild barley accessions, XZ72 and XZ95 were micro-
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malted using Joe White Malting System (Adelaide, SA, Australia). The malting procedures
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were as below: (1) Steeping stage: 5–8–8–12–4–5–2 h (wet–dry–wet–dry–wet–dry–wet); (2)
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Germination stage: 96 h at 16 °C. GA (0.5 mg/l) or ABA (0.5 mg/l) was added at the last step
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of wet stage during steeping. Seed samples were collected after steeping stage and 24 h, 48 h,
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72 h, 96 h after germination.
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2.2. Determination of enzyme activity and β-glucan content. The activities of α-amylase,
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β-amylase, β-glucanase and limit dextrinase, and β-glucan content were measured using
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Megazyme assay kits (Megazyme International, Bray, Ireland), according to the
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manufacture’s manual instruction.
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2.3. Protein extraction and digestion. Frozen barley grains after steeping and 96 h after
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germination were ground under liquid nitrogen and extracted in lysis buffer (8 M urea, 1%
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Triton-100, 65 mM dithiothreitol (DTT) and 0.1% Protease inhibitor Cocktail (Sigma)).26
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After centrifugation at 20000 g at 4 °C for 10 min, the supernatant was collected and
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precipitated with cold trichloroacetic acid (TCA) for 2h at -20 °C. The supernatant was
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discarded after centrifugation at 4 °C for 10 min. The final pellet was dissolved in 8 M urea,
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100 mM triethylammonium bicarbonate (TEAB, pH 8.0) buffer before treatment of 10 mM
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DTT to reduce the protein and 20 mM indole-3-acetic acid (IAA) at room temperature in
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darkness for alkylation. The protein solution was then diluted to less than 2 M urea with 100
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mM TEAB. Trypsin was added to a final trypsin/protein mass ratio of 1:50 and left overnight,
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followed by an additional digest at 1:100 ratio for 4 h. 27
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2.4. iTRAQ labelling. The above protein solution was attached to a Strata×C18 SPE
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column for desalination and were then vacuum-dried. The tryptic peptides were dissolved in
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0.5 M TEAB buffer and incubated with 8-plex iTRAQ labelling kit for 2 h at room
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temperature. One hundred micrograms proteins of XZ72 and XZ95 collected after steeping
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and germination were labelled with iTRAQ reporters.
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2.5. Peptide fractionation and quantitative proteomic analysis by LC-MS/MS. The
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labelled samples were fractionated by high pH reverse-phase HPLC system connected to an
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Agilent 300 Extend C18 column (5 µm particles, 4.6 mm internal diameter, 250 mm length).
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The peptides were dissolved in 0.1% formic acid and then loaded onto a reversed-phase pre-
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column (Acclaim PepMan 100, Thermo Scientific). A reversed-phase analytical column
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(Acclaim PepMan RSLC, Thermo Scientific) was used to perform peptide separation. The
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workflow for gradient separation was set as follows: from 6% to 22% solvent B (0.1% FA in
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98% ACN) over 24 min, 22% to 36% in 8 min and 80% in 4 min and then maintained at 80%
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for another 4 min at a constant flow rate of 280 nl/min on an EASY-nLC 1000 HPLC system.
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The peptides were analysed by Q ExactiveTM Plus hybrid quadrupole-Orbitrap mass
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spectrometer (Thermo Fisher Scientific).
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The resolution of Orbitrap detection was set to 70,000. The voltage for electrospray was set
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at 2.0 kV. Automatic gain control (AGC) was used to prevent overfilling of the ion trap, and
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it was set to 500,000 ions. The mass spectra scan range was 350 to 1800. Dynamic exclusion
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duration in the MS survey scan was 30.0 s and 20 most intensive signals were fragmented by
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MS/MS.
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2.6. Data analysis. The raw MS/MS data was processed by Mascot search engine (version
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2.3.0, Matrix Science, London, U.K.). All searches were performed by Hordeum vulgare
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protein database (http://www.uniprot.org/taxonomy/?query=Hordeum+vulgare&sort=score)
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which contains 73,385 sequences. Carbamidomethyl, iTRAQ-8 plex (N-term) and iTRAQ-8
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plex (K) were set as fixed modification, and oxidation on Met was chosen as variable
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modifications. Mass error for precursor ions and fragment ions were set to 10 ppm and 0.02
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Da, respectively. Cleaving enzyme which was represented by trypsin/P was allowed up to 2
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missing cleavage. The filters for protein identification were: significance threshold of P
